In the United States and elsewhere in the technologically developed world, it is customary and often legally mandated, to assess the capacity of consumer products such as shampoos, detergents, cosmetics, or other materials which are likely to be handled by the general public without eye protection, to cause temporary or permanent damage to the human eye. Because of the sensitivity and criticality of ocular tissue, it is clearly desirable and necessary to provide adequate labeling in the case of mild and temporary eye irritants or even to restrict the commercialization of compositions which contain components extremely damaging to the eye. It is estimated that more than a million tests are performed annually in the United States to examine various cosmetic and household produts for their potential harmfulness to the human eye. As in any instance where large numbers of tests need to be performed to screen for a particular property, it is desirable to have a testing procedure which is rapid, inexpensive, and reliable. Certainly an in vitro test would be most desirable.
Such an in vitro test is not currently available for substances which might irritate the eye. The most commonly utilized current screening procedure is an in vivo one, the Draize rabbit-eye test (Draize, J. H., et al J Pharmacol Exptl Therap (1944) 82:377). The disadvantages of this test are legion. First, as it is an in vivo procedure it necessarily involves some degree of maltreatment of test animals, and considerable expense. Secondly, it is lengthy. The procedure consists of placing the substance to be tested into the eyes of 3-9 albino rabbits and scoring the degree of irritation on the conjunctiva, cornea, and iris after periods of 3-21 days. The scoring of the results is, of course, subjective to some degree. The individuality of the test animals makes the results inherently unreproducible. Many attempts have been made to refine and improve the Draize test and to minimize its disadvantages (see, for example, Batista, S. P., et al, Soc Cosmet Chemists (1965) 16:119; Kay, J. H., et al, Am Perfumer Cosmetics (1965) 80:61; Gaunt, I. F., et al, J Soc Cosmet Chemists (1964) 15:209). However, the inherent deficiencies of this testing approach make impossible the attainment of the objective of a cheap, reliable, reproducible and predictive test for eye irritating properties.
As in all animal tests, the correlation with effects of the materials in human subjects is imperfect; however, the Draize test has become sufficiently well recognized, that a more straightforward test which produces results identical to those of the Draize would represent an improvement in the state of the art, and correlation with Draize results can reasonably be used to evaluate such a test.
The clear desirability of an in vitro procedure has led a number of researchers to devise tests involving cell cultures as opposed to whole animals. For example, the method of Ferguson, T. F. M., et al, Food and Cosmet Toxicol (1974) 12:359 employs cultured mouse fibroblast cells and uses the ability of a material to inhibit the uptake of tritiated uridine by these cells as a measure of its eye irritation capacity. The method of Stark, L., et al, Chemical Week (1983) May 26:27 uses a procedure whereby mouse cell cultures are exposed to irritants, and the fluids from such cultures evaluated for their effect on the migration of macrophages. The method of Char Jumblatt, M., Vision Res (1981) 21:45 uses the level of plasminogen activator in rabbit corneal cells in culture as a measure of the eye-irritating capability of a substance. Finally, inhibition of culture growth in fibroblast or HeLa cells was used as a criterion by Litterst, C. L., et al, Arch Environ Health (1971) 22:454. Two additional methods employ mouse or rabbit ileum and assess, respectively, the penetration capacity of the substance to be tested (Muir, C. K., Toxicology Letters (1983) 19:309) and the response of a chorioallantroic membrane of chick embryos (Leighton, J., et al, Proc of the Symposium, Product Safety Evaluation, A. M. Goldberg, Editor, Mary Ann Liebert Publications, New York (1983)).
All of the foregoing in vitro methods require either living cells in tissue culture or isolated membranes in tissue culture . All suffer from the disadvantages of lack of reproducibility, lack of objectivity, and lack of perfect correlation with the property desired to be measured, as does the Draize method. While these procedures provide an alternative to the strictly in vivo approach of Draize, they do not achieve the simplicity and standardization one expects from testing based on chemical reagents which can be manufactured and stored, and reproduced exactly. The method of the present invention offers such a test. It provides a standard, quick, reproducible, objective measure of the capacity of any material to cause irritation in the human eye. It does not involve use of small animals, and does not require the expense of maintaining, caging, and feeding facilities for them.